Plant Immunity against Tobamoviruses.

Tobamoviruses are a group of plant viruses that pose a significant threat to agricultural crops worldwide. In this review, we focus on plant immunity against tobamoviruses, including pattern-triggered immunity (PTI), effector-triggered immunity (ETI), the RNA-targeting pathway, phytohormones, reactive oxygen species (ROS), and autophagy. Further, we highlight the genetic resources for resistance against tobamoviruses in plant breeding and discuss future directions on plant protection against tobamoviruses.

Viral Threats to Fruit and Vegetable Crops in the Caribbean.

Viruses pose major global challenges to crop production as infections reduce the yield and quality of harvested products, hinder germplasm exchange, increase financial inputs, and threaten food security. Small island or archipelago habitat conditions such as those in the Caribbean are particularly susceptible as the region is characterized by high rainfall and uniform, warm temperatures throughout the year. Moreover, Caribbean islands are continuously exposed to disease risks because of their location at the intersection of transcontinental trade between North and South America and their role as central hubs for regional and global agricultural commodity trade. This review provides a summary of virus disease epidemics that originated in the Caribbean and those that were introduced and spread throughout the islands. Epidemic-associated factors that impact disease development are also discussed. Understanding virus disease epidemiology, adoption of new diagnostic technologies, implementation of biosafety protocols, and widespread acceptance of biotechnology solutions to counter the effects of cultivar susceptibility remain important challenges to the region. Effective integrated disease management requires a comprehensive approach that should include upgraded phytosanitary measures and continuous surveillance with rapid and appropriate responses.

Antibody-based defense may protect plants from disease.

A "creative" strategy to keep crops healthy borrows key pathogen detectors from the animal immune system.

Genetic Differentiation and Migration Fluxes of Viruses from Melon Crops and Crop Edge Weeds.

Weeds surrounding crops may act as alternative hosts, playing important epidemiological roles as virus reservoirs and impacting virus evolution. We used high-throughput sequencing to identify viruses in Spanish melon crops and plants belonging to three pluriannual weed species, Ecballium elaterium, Malva sylvestris, and Solanum nigrum, sampled at the edges of the crops. Melon and E. elaterium, both belonging to the family Cucurbitaceae, shared three virus species, whereas there was no virus species overlap between melon and the other two weeds. The diversity of cucurbit aphid-borne yellows virus (CABYV) and tomato leaf curl New Delhi virus (ToLCNDV), both in melon and E. elaterium, was further studied by amplicon sequencing. Phylogenetic and population genetics analyses showed that the CABYV population was structured by the host, identifying three sites in the CABYV RNA-dependent RNA polymerase under positive selection, perhaps reflecting host adaptation. The ToLCNDV population was much less diverse than the CABYV one, likely as a consequence of the relatively recent introduction of ToLCNDV in Spain. In spite of its low diversity, we identified geographical but no host differentiation for ToLCNDV. Potential virus migration fluxes between E. elaterium and melon plants were also analyzed. For CABYV, no evidence of migration between the populations of the two hosts was found, whereas important fluxes were identified between geographically distant subpopulations for each host. For ToLCNDV, in contrast, evidence of migration from melon to E. elaterium was found, but not the other way around. IMPORTANCE It has been reported that about half of the emerging diseases affecting plants are caused by viruses. Alternative hosts often play critical roles in virus emergence as virus reservoirs, bridging host species that are otherwise unconnected and/or favoring virus diversification. In spite of this, the viromes of potential alternative hosts remain largely unexplored. In the case of crops, pluriannual weeds at the crop edges may play these roles. Here, we took advantage of the power of high-throughput sequencing to characterize the viromes of three weed species frequently found at the edges of melon crops. We identified three viruses shared by melon and the cucurbit weed, with two of them being epidemiologically relevant for melon crops. Further genetic analyses showed that these two viruses had contrasting patterns of diversification and migration, providing an interesting example on the role that weeds may play in the ecology and evolution of viruses affecting crops.

One-Enzyme RTX-PCR for the Detection of RNA Viruses from Multiple Virus Genera and Crop Plants.

Reverse transcription PCR (RT-PCR) is a popular method for detecting RNA viruses in plants. RT-PCR is usually performed in a classical two-step procedure: in the first step, cDNA is synthesized by reverse transcriptase (RT), followed by PCR amplification by a thermostable polymerase in a separate tube in the second step. However, one-step kits containing multiple enzymes optimized for RT and PCR amplification in a single tube can also be used. Here, we describe an RT-PCR single-enzyme assay based on an RTX DNA polymerase that has both RT and polymerase activities. The expression plasmid pET_RTX_(exo-) was transferred to various E. coli genotypes that either compensated for codon bias (Rosetta-gami 2) or contained additional chaperones to promote solubility (BL21 (DE3) with plasmids pKJE8 or pTf2). The RTX enzyme was then purified and used for the RT-PCR assay. Several purified plant viruses (TMV, PVX, and PVY) were used to determine the efficiency of the assay compared to a commercial one-step RT-PCR kit. The RT-PCR assay with the RTX enzyme was validated for the detection of viruses from different genera using both total RNA and crude sap from infected plants. The detection endpoint of RTX-PCR for purified TMV was estimated to be approximately 0.01 pg of the whole virus per 25 µL reaction, corresponding to 6 virus particles/µL. Interestingly, the endpoint for detection of TMV from crude sap was also 0.01 pg per reaction in simulated crude plant extracts. The longest RNA fragment that could be amplified in a one-tube arrangement was 2379 bp long. The longest DNA fragment that could be amplified during a 10s extension was 6899 bp long. In total, we were able to detect 13 viruses from 11 genera using RTX-PCR. For each virus, two to three specific fragments were amplified. The RT-PCR assay using the RTX enzyme described here is a very robust, inexpensive, rapid, easy to perform, and sensitive single-enzyme assay for the detection of plant viruses.

A cluster of atypical resistance genes in soybean confers broad-spectrum antiviral activity.

Soybean mosaic virus (SMV) is a severe soybean (Glycine max) pathogen. Here we characterize a soybean SMV resistance cluster (SRC) that comprises five resistance (R) genes. SRC1 encodes a Toll/interleukin-1 receptor and nucleotide-binding site (TIR-NBS [TN]) protein, SRC4 and SRC6 encode TIR proteins with a short EF-hand domain, while SRC7 and SRC8 encode TNX proteins with a noncanonical basic secretory protein (BSP) domain at their C-termini. We mainly studied SRC7, which contains a noncanonical BSP domain and gave full resistance to SMV. SRC7 possessed broad-spectrum antiviral activity toward several plant viruses including SMV, plum pox virus, potato virus Y, and tobacco mosaic virus. The TIR domain alone was both necessary and sufficient for SRC7 immune signaling, while the NBS domain enhanced its activity. Nuclear oligomerization via the interactions of both TIR and NBS domains was essential for SRC7 function. SRC7 expression was transcriptionally inducible by SMV infection and salicylic acid (SA) treatment, and SA was required for SRC7 triggered virus resistance. SRC7 expression was posttranscriptionally regulated by miR1510a and miR2109, and the SRC7-miR1510a/miR2109 regulatory network appeared to contribute to SMV-soybean interactions in both resistant and susceptible soybean cultivars. In summary, we report a soybean R gene cluster centered by SRC7 that is regulated at both transcriptional and posttranscriptional levels, possesses a yet uncharacterized BSP domain, and has broad-spectrum antiviral activities. The SRC cluster is special as it harbors several functional R genes encoding atypical TIR-NBS-LRR (TNL) type R proteins, highlighting its importance in SMV-soybean interaction and plant immunity.

MiR398-regulated antioxidants contribute to Bamboo mosaic virus accumulation and symptom manifestation.

Virus infections that cause mosaic or mottling in leaves commonly also induce increased levels of reactive oxygen species (ROS). However, how ROS contributes to symptoms is less well documented. Bamboo mosaic virus (BaMV) causes chlorotic mosaic symptoms in both Brachypodium distachyon and Nicotiana benthamiana. The BaMV △CPN35 mutant with an N-terminal deletion of its coat protein gene exhibits asymptomatic infection independently of virus titer. Histochemical staining of ROS in mock-, BaMV-, and BaMV△CPN35-infected leaves revealed that hydrogen peroxide (H2O2) accumulated solely in BaMV-induced chlorotic spots. Moreover, exogenous H2O2 treatment enhanced yellowish chlorosis in BaMV-infected leaves. Both BaMV and BaMV△CPN35 infection could induce the expression of Cu/Zu superoxide dismutase (CSD) antioxidants at messenger RNA and protein level. However, BaMV triggered the abundant accumulation of full-length NbCSD2 preprotein (prNbCSD2, without transit peptide cleavage), whereas BaMV△CPN35 induced a truncated prNbCSD2. Confocal microscopy showed that majority of NbCSD2-green fluorescent protein (GFP) predominantly localized in the cytosol upon BaMV infection, but BaMV△CPN35 infection tended to cause NbCSD2-GFP to remain in chloroplasts. By 5'-RNA ligase-mediated rapid amplification of cDNA ends, we validated CSDs are the targets of miR398 in vivo. Furthermore, BaMV infection increased the level of miR398, while the level of BaMV titer was regulated positively by miR398 but negatively by CSD2. In contrast, overexpression of cytosolic form NbCSD2, impairing the transport into chloroplasts, greatly enhanced BaMV accumulation. Taken together, our results indicate that induction of miR398 by BaMV infection may facilitate viral titer accumulation, and cytosolic prNbCSD2 induction may contribute to H2O2 accumulation, resulting in the development of BaMV chlorotic symptoms in plants.

Integrating High throughput Sequencing into Survey Design Reveals Turnip Yellows Virus and Soybean Dwarf Virus in Pea (Pisum Sativum) in the United Kingdom.

There is only limited knowledge of the presence and incidence of viruses in peas within the United Kingdom, therefore high-throughput sequencing (HTS) in combination with a bulk sampling strategy and targeted testing was used to determine the virome in cultivated pea crops. Bulks of 120 leaves collected from twenty fields from around the UK were initially tested by HTS, and presence and incidence of virus was then determined using specific real-time reverse-transcription PCR assays by testing smaller mixed-bulk size samples. This study presents the first finding of turnip yellows virus (TuYV) in peas in the UK and the first finding of soybean dwarf virus (SbDV) in the UK. While TuYV was not previously known to be present in UK peas, it was found in 13 of the 20 sites tested and was present at incidences up to 100%. Pea enation mosaic virus-1, pea enation mosaic virus-2, pea seed-borne mosaic virus, bean yellow mosaic virus, pea enation mosaic virus satellite RNA and turnip yellows virus associated RNA were also identified by HTS. Additionally, a subset of bulked samples were re-sequenced at greater depth to ascertain whether the relatively low depth of sequencing had missed any infections. In each case the same viruses were identified as had been identified using the lower sequencing depth. Sequencing of an isolate of pea seed-borne mosaic virus from 2007 also revealed the presence of TuYV and SbDV, showing that both viruses have been present in the UK for at least a decade, and represents the earliest whole genome of SbDV from Europe. This study demonstrates the potential of HTS to be used as a surveillance tool, or for crop-specific field survey, using a bulk sampling strategy combined with HTS and targeted diagnostics to indicate both presence and incidence of viruses in a crop.

Modelling interference between vectors of non-persistently transmitted plant viruses to identify effective control strategies.

Aphids are the primary vector of plant viruses. Transient aphids, which probe several plants per day, are considered to be the principal vectors of non-persistently transmitted (NPT) viruses. However, resident aphids, which can complete their life cycle on a single host and are affected by agronomic practices, can transmit NPT viruses as well. Moreover, they can interfere both directly and indirectly with transient aphids, eventually shaping plant disease dynamics. By means of an epidemiological model, originally accounting for ecological principles and agronomic practices, we explore the consequences of fertilization and irrigation, pesticide deployment and roguing of infected plants on the spread of viral diseases in crops. Our results indicate that the spread of NPT viruses can be i) both reduced or increased by fertilization and irrigation, depending on whether the interference is direct or indirect; ii) counter-intuitively increased by pesticide application and iii) reduced by roguing infected plants. We show that a better understanding of vectors' interactions would enhance our understanding of disease transmission, supporting the development of disease management strategies.

Spatial parameters associated with the risk of banana bunchy top disease in smallholder systems.

The Banana Bunchy Top Disease (BBTD), caused by the Banana Bunchy Top Virus (BBTV) is the most important and devastating in many tropical countries. BBTD epidemiology has been little studied, mixed landscape smallholder systems. The relative risks associated with this disease vary between geographical areas and landscapes. This work analyzed the management and vegetation conditions in smallholder gardens to assess the factors linked to landscape-level BBTV transmission and management. Mapping was done in this study area which is in a BBTD-endemic region, involving farmers actively managing the disease, but with household-level decision making. A spatial scanning statistic was used to detect and identify spatial groups at the 5% significance threshold, and a Poisson regression model was used to explore propagation vectors and the effect of surrounding vegetation and crop diversity. Spatial groups with high relative risk were identified in three communities, Dangbo, Houéyogbé, and Adjarra. Significant associations emerged between the BBTD prevalence and some crop diversity, seed systems, and BBTD management linked factors. The identified factors form important candidate management options for the detailed assessment of landscape-scale BBTD management in smallholder communities.

California TRV-based VIGS vectors mediate gene silencing at elevated temperatures but with greater growth stunting.

BACKGROUND: Tobacco rattle virus (TRV) based virus-induced gene silencing (VIGS), a widely used functional genomics tool, requires growth temperatures typically lower than those of the plant's native environment. Enabling VIGS under native conditions in the field according to applicable safety regulations could be a revolutionary advance for ecological research. RESULTS: Here, we report the development of an enhanced thermal tolerant VIGS vector system based on a TRV California isolate. cDNA clones representing the whole viral genome were sequenced and used to construct separate binary plant transformation vectors for functional elements of RNA1 (6765 nt) and RNA2 (3682 nt). VIGS of target genes was induced by transient transformation of the host plant with both vectors or by treating the host plant with sap from already VIGS induced plants. In Nicotiana attenuata the silencing efficiency of the PDS (phytoene desaturase) gene was 90% at 28 °C and 78% at 30 °C. Silencing at these temperatures was more prominent and durable than silencing induced by the widely used TRV PpK20-based pBINTRA6/pTV00 system, but was associated with a viral phenotype. Differences in the suppressor protein and RNA dependent RNA polymerase sequences between the TRV California isolate and PpK20 may be the reason for their different thermal tolerance. CONCLUSIONS: The new TRV California-based VIGS vectors induce gene silencing in Nicotiana attenuata at higher temperatures than the existing pBINTRA6/pTV00 vector system, but cause greater growth defects. The new vector system opens up an avenue to study genes functions in planta under field conditions.

Identification of novel QTL contributing to barley yellow mosaic resistance in wild barley (Hordeum vulgare spp. spontaneum).

BACKGROUND: Barley yellow mosaic disease (BYMD) caused by Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV) seriously threatens the production of winter barley. Cultivating and promoting varieties that carry disease-resistant genes is one of the most powerful ways to minimize the disease's effect on yield. However, as the BYMD virus mutates rapidly, resistance conferred by the two cloned R genes to the virus had been overcome by new virus strains. There is an urgent need for novel resistance genes in barley that convey sustainable resistance to newly emerging virus strains causing BYMD. RESULTS: A doubled haploid (DH) population derived from a cross of SRY01 (BYMD resistant wild barley) and Gairdner (BYMD susceptible barley cultivar) was used to explore for QTL of resistance to BYMD in barley. A total of six quantitative trait loci (qRYM-1H, qRYM-2Ha, qRYM-2Hb, qRYM-3H, qRYM-5H, and qRYM-7H) related to BYMD resistance were detected, which were located on chromosomes 1H, 2H, 3H, 5H, and 7H. Both qRYM-1H and qRYM-2Ha were detected in all environments. qRYM-1H was found to be overlapped with rym7, a known R gene to the disease, whereas qRYM-2Ha is a novel QTL on chromosome 2H originated from SRY01, explaining phenotypic variation from 9.8 to 17.8%. The closely linked InDel markers for qRYM-2Ha were developed which could be used for marker-assisted selection in barley breeding. qRYM-2Hb and qRYM-3H were stable QTL for specific resistance to Yancheng and Yangzhou virus strains, respectively. qRYM-5H and qRYM-7H identified in Yangzhou were originated from Gairdner. CONCLUSIONS: Our work is focusing on a virus disease (barley yellow mosaic) of barley. It is the first report on BYMD-resistant QTL from wild barley accessions. One novel major QTL (qRYM-2Ha) for the resistance was detected. The consistently detected new genes will potentially serve as novel sources for achieving pre-breeding barley materials with resistance to BYMD.

A rapid, simple, and highly efficient method for VIGS and in vitro-inoculation of plant virus by INABS applied to crops that develop axillary buds and can survive from cuttings.

BACKGROUND: Virus-induced gene silencing (VIGS) is one of the most convenient and powerful methods of reverse genetics. In vitro-inoculation of plant virus is an important method for studying the interactions between viruses and plants. Agrobacterium-based infiltration has been widely adopted as a tool for VIGS and in vitro-inoculation of plant virus. Most agrobacterium-based infiltration methods applied to VIGS and virus inoculation have the characteristics of low transformation efficiencies, long plant growth time, large amounts of plant tissue, large test spaces, and complex preparation procedures. Therefore, a rapid, simple, economical, and highly efficient VIGS and virus inoculation method is in need. Previous studies have shown that the selection of suitable plant tissues and inoculation sites is the key to successful infection. RESULTS: In this study, Tobacco rattle virus (TRV) mediated VIGS and Tomato yellow leaf curl virus (TYLCV) for virus inoculation were developed in tomato plants based on the agrobacterium tumefaciens-based infiltration by injection of the no-apical-bud stem section (INABS). The no-apical-bud stem section had a "Y- type" asymmetric structure and contained an axillary bud that was about 1-3 cm in length. This protocol provides high transformation (56.7%) and inoculation efficiency (68.3%), which generates VIGS transformants or diseased plants in a very short period (8 dpi). Moreover, it greatly reduces the required experimental space. This method will facilitate functional genomic studies and large-scale disease resistance screening. CONCLUSIONS: Overall, a rapid, simple, and highly efficient method for VIGS and virus inoculation by INABS was developed in tomato. It was reasonable to believe that it can be used as a reference for the other virus inoculation methods and for the application of VIGS to other crops (such as sweet potato, potato, cassava and tobacco) that develop axillary buds and can survive from cuttings.

Virus Diseases of Cereal and Oilseed Crops in Australia: Current Position and Future Challenges.

This review summarizes research on virus diseases of cereals and oilseeds in Australia since the 1950s. All viruses known to infect the diverse range of cereal and oilseed crops grown in the continent's temperate, Mediterranean, subtropical and tropical cropping regions are included. Viruses that occur commonly and have potential to cause the greatest seed yield and quality losses are described in detail, focusing on their biology, epidemiology and management. These are: barley yellow dwarf virus, cereal yellow dwarf virus and wheat streak mosaic virus in wheat, barley, oats, triticale and rye; Johnsongrass mosaic virus in sorghum, maize, sweet corn and pearl millet; turnip yellows virus and turnip mosaic virus in canola and Indian mustard; tobacco streak virus in sunflower; and cotton bunchy top virus in cotton. The currently less important viruses covered number nine infecting nine cereal crops and 14 infecting eight oilseed crops (none recorded for rice or linseed). Brief background information on the scope of the Australian cereal and oilseed industries, virus epidemiology and management and yield loss quantification is provided. Major future threats to managing virus diseases effectively include damaging viruses and virus vector species spreading from elsewhere, the increasing spectrum of insecticide resistance in insect and mite vectors, resistance-breaking virus strains, changes in epidemiology, virus and vectors impacts arising from climate instability and extreme weather events, and insufficient industry awareness of virus diseases. The pressing need for more resources to focus on addressing these threats is emphasized and recommendations over future research priorities provided.

Development of a Csy4-processed guide RNA delivery system with soybean-infecting virus ALSV for genome editing.

BACKGROUND: A key issue for implementation of CRISPR-Cas9 genome editing for plant trait improvement and gene function analysis is to efficiently deliver the components, including guide RNAs (gRNAs) and Cas9, into plants. Plant virus-based gRNA delivery strategy has proven to be an important tool for genome editing. However, its application in soybean which is an important crop has not been reported yet. ALSV (apple latent spherical virus) is highly infectious virus and could be explored for delivering elements for genome editing. RESULTS: To develop a ALSV-based gRNA delivery system, the Cas9-based Csy4-processed ALSV Carry (CCAC) system was developed. In this system, we engineered the soybean-infecting ALSV to carry and deliver gRNA(s). The endoribonuclease Csy4 effectively releases gRNAs that function efficiently in Cas9-mediated genome editing. Genome editing of endogenous phytoene desaturase (PDS) loci and exogenous 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) sequence in Nicotiana. benthamiana (N. benthamiana) through CCAC was confirmed using Sanger sequencing. Furthermore, CCAC-induced mutagenesis in two soybean endogenous GW2 paralogs was detected. CONCLUSIONS: With the aid of the CCAC system, the target-specific gRNA(s) can be easily manipulated and efficiently delivered into soybean plant cells by viral infection. This is the first virus-based gRNA delivery system for soybean for genome editing and can be used for gene function study and trait improvement.

Investigations on annual spreading of viruses infecting cucurbit crops in Uttar Pradesh State, India.

During 2018 an intensive study was conducted to determine the viruses associated with cucurbitaceous crops in nine agroclimatic zones of the state of Uttar Pradesh, India. Total of 563 samples collected and analysed across 14 different cucurbitaceous crops. The results showed the dominance of Begomovirus (93%) followed by Potyvirus (46%), cucumber green mottle mosaic virus (CGMMV-39%), Polerovirus (9%), cucumber mosaic virus (CMV-2%) and Orthotospovirus (2%). Nearly 65% of samples were co-infected with more than one virus. Additionally, host range expansion of CMV, CGMMV and polerovirus was also observed on cucurbit crops. A new potyvirus species, zucchini tigre mosaic virus, earlier not documented from India has also been identified on five crops during the study. Risk map generated using ArcGIS for virus disease incidence predicted the virus severity in unexplored areas. The distribution pattern of different cucurbit viruses throughout Uttar Pradesh will help identify the hot spots for viruses and will facilitate to devise efficient and eco-friendly integrated management strategies for the mitigation of viruses infecting cucurbit crops. Molecular diversity and evolutionary relationship of the virus isolates infecting cucurbits in Uttar Pradesh with previously reported strains were understood from the phylogenetic analysis. Diverse virus infections observed in the Eastern Plain zone, Central zone and North-Eastern Plain zone indicate an alarming situation for the cultivation of cucurbits in the foreseeable future.

Collection, genotyping and virus elimination of cassava landraces from Tanzania and documentation of farmer knowledge.

Cassava (Manihot esculenta Crantz.) has been a vital staple and food security crop in Tanzania for several centuries, and it is likely that its resilience will play a key role in mitigating livelihood insecurities arising from climate change. The sector is dominated by smallholder farmers growing traditional landrace varieties. A recent surge in virus diseases and awareness in the commercial potential of cassava has prompted a drive to disseminate improved varieties in the country. These factors however also threaten the existence of landraces and associated farmer knowledge. It is important that the landraces are conserved and utilized as the adaptive gene complexes they harbor can drive breeding for improved varieties that meet agro-ecological adaptation as well as farmer and consumer needs, thereby improving adoption rates. Here we report on cassava germplasm collection missions and documentation of farmer knowledge in seven zones of Tanzania. A total of 277 unique landraces are identified through high-density genotyping. The large number of landraces is attributable to a mixed clonal/sexual reproductive system in which the soil seed bank and incorporation of seedlings plays an important role. A striking divergence in genetic relationships between the coastal regions and western regions is evident and explained by (i) independent introductions of cassava into the country, (ii) adaptation to prevailing agro-ecological conditions and (iii) farmer selections according to the intended use or market demands. The main uses of cassava with different product profiles are evident, including fresh consumption, flour production, dual purpose incorporating both these uses and longer-term food security. Each of these products have different trait requirements. Individual landraces were not widely distributed across the country with limited farmer-to-farmer diffusion with implications for seed systems.

Detection of plant virus particles with a capacitive field-effect sensor.

Plant viruses are major contributors to crop losses and induce high economic costs worldwide. For reliable, on-site and early detection of plant viral diseases, portable biosensors are of great interest. In this study, a field-effect SiO2-gate electrolyte-insulator-semiconductor (EIS) sensor was utilized for the label-free electrostatic detection of tobacco mosaic virus (TMV) particles as a model plant pathogen. The capacitive EIS sensor has been characterized regarding its TMV sensitivity by means of constant-capacitance method. The EIS sensor was able to detect biotinylated TMV particles from a solution with a TMV concentration as low as 0.025 nM. A good correlation between the registered EIS sensor signal and the density of adsorbed TMV particles assessed from scanning electron microscopy images of the SiO2-gate chip surface was observed. Additionally, the isoelectric point of the biotinylated TMV particles was determined via zeta potential measurements and the influence of ionic strength of the measurement solution on the TMV-modified EIS sensor signal has been studied.

Use of Molecular Markers for Doubled Haploid Technology: From Academia to Plant Breeding Companies.

Molecular markers are employed for doubled haploid (DH) technology by researchers and applied plant breeders in many crops. In the 1990s, isozymes and RFLPs were commonly used marker technologies to characterize DHs and were later replaced by PCR- based markers (e.g., RAPDs, AFLPs, ISSRs, SSRs) and today by SNPs. Markers are used for multiple purposes in DH production, that is, for the study of genes underlying haploid induction and confirming homozygous plants of gametophytic origin. Furthermore, they are tools for investigating segregation in DH populations and for mapping simple and complex traits using DHs. The deployment of DHs and markers for developing trait-linked markers are demonstrated with examples from rapeseed, wheat, and barley. Marker development for resistance to viruses derived from genetic resources and their use in, for example, pyramiding of resistance genes, are given as an example for the combination of DH-technology and marker development in research. Today, marker systems amenable to automation are frequently used in applied plant breeding. Practical examples are given from Lantmännen (LM) ( https://Lantmannen.com ) using large-scale genotyping for variety development based on SSRs and SNPs.